Endostatin Inhibits Microvessel Formation in the ex Vivo Rat Aortic Ring Angiogenesis Assay

Krüger, Erwin A.; Duray, Paul H.; Tsokos, Maria; Venzon, David; Libutti, Steven K.; Dixon, Shannon C.; Rudek, Michelle A.; Pluda, James M.; Allegra, Carmen J.; Figg, William D.

doi:10.1006/bbrc.1999.2018

Cited by 75 publications

(51 citation statements)

References 17 publications

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“…This is in agreement with other recent studies in which the angiostatic effect of endostatin on endothelial cells was shown in vitro (Dhanabal et al, 1999a(Dhanabal et al, , 1999bYamaguchi et al, 1999) and in vivo (Dhanabal et al, 1999a;O'Reilly et al, 1997). In contrast to recent observations by Kruger et al (2000) who used the human saphenous vein model and the rat aortic ring model, we did not detect a species difference in the antiangiogenic activity of endostatin. It should be noted that endothelial cells surrounding E17 metatarsals positively stained for collagen XVIII (own unpublished results), the endogenous source of mouse endostatin, which might explain the relatively high levels (5 g/ml) of this agent needed to block the angiogenic response as compared with other in vitro studies (Taddei et al, 1999;Yamaguchi et al, 1999).…”

Section: Deckers Et Alsupporting

confidence: 93%

Effect of Angiogenic and Antiangiogenic Compounds on the Outgrowth of Capillary Structures from Fetal Mouse Bone Explants

Deckers

Pluijm

Dooijewaard

et al. 2001

Laboratory Investigation

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SUMMARY:Fetal mouse metatarsals are well-known models to study cartilage differentiation and osteoclastic resorption. We show here the outgrowth of PECAM-1 positive tubelike structures from the bone rudiments. This feature can be used to study angiogenesis in vitro. The area of outgrowth significantly increased with culture time, as shown by computerized image analysis of PECAM-1 positive tubelike structures. Treatment with recombinant human vascular endothelial growth factor (rhVEGF-A) stimulated the formation of tubelike structures. Treatment of explants with the angiogenesis inhibitor endostatin, the chemokine IP-10, and the thalidomide derivative phatolyl glutamic acid (PG-acid) resulted in an inhibition of the formation of PECAM-1 positive tubelike structures of 48.8% (Ϯ 4%), 50.2% (Ϯ12%), and 80.8% (Ϯ3%), respectively. Outgrowth of tubelike structures was partly dependent on endogenous VEGF-A because treatment with anti-mVEGF-A and truncated VEGF receptor 1 (soluble fms-like tyrosine kinase 1, sFlt1) strongly inhibited the formation of tubelike structures 74% (Ϯ 4%) and 38% (Ϯ 5%), respectively. Neither onset of tube formation nor total area of tubelike structures were changed when metatarsals were cultured on a fibrin gel or collagen type I gel. Tube formation required activation of matrix metalloproteinases because treatment of the bones with an inhibitor of matrix metalloproteinases completely inhibited migration and tube formation, whereas treatment with an inhibitor of plasmin had no effect. In conclusion, we describe a new in vitro model to study angiogenesis that can be used to test the angiogenic or antiangiogenic potential of novel test compounds that also combines the multicellularity of in vivo assays with the accessibility and flexibility of in vitro assays. (Lab Invest 2001, 81:5-15).

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Section: Deckers Et Alsupporting

confidence: 93%

Effect of Angiogenic and Antiangiogenic Compounds on the Outgrowth of Capillary Structures from Fetal Mouse Bone Explants

Deckers

Pluijm

Dooijewaard

et al. 2001

Laboratory Investigation

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“…[3][4][5][12][13][14][15]26 In this study, we describe a reproducible system for investigating the angiogenic cascade by controlling experimental variables and precisely quantifying angiogenic outgrowth. We developed a human angiogenesis model, in which umbilical artery rings embedded in BME produced capillarylike structures, recapitulating different steps of angiogenesis, including EC sprouting, migration, and differentiation into capillaries.…”

Section: Discussionmentioning

confidence: 99%

“…[8][9][10][11] However, rat and mAR models display certain disadvantages: a high age-and strain-dependent variability of assays, 12 as well as variations owing to the region of the explanted aorta 13 ; autonomous angiogenic outgrowth, limiting studies on proangiogenic factors 14 ; the necessity to sacrifice animals; and the failure to detect species-specific antiangiogenic compounds in preclinical screening assays. 15 In this study, we developed an ex vivo three-dimensional (3D) assay of sprouting angiogenesis with arterial explants from human umbilical cords. This assay can be exploited for antiangiogenic drug screening and, when coupled with lentiviral-mediated gene expression, for direct gene function analysis on developing human vessels.…”

Section: Introductionmentioning

confidence: 99%

Modeling human tumor angiogenesis in a three-dimensional culture system

Seano

Chiaverina

Gagliardi

et al. 2013

Blood

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Key Points• Human arterial ring assay is an innovative system for the three-dimensional study of tumor angiogenesis.• This assay can be exploited for antiangiogenic drug screening and gene function analysis on human vessels.The intrinsic complexity of the process of vessel formation limits the efficacy of cellular assays for elucidation of its molecular and pharmacologic mechanisms. We developed an ex vivo three-dimensional (3D) assay of sprouting angiogenesis with arterial explants from human umbilical cords. In this assay, human arterial rings were embedded in basement membrane extract gel, leading to a network of capillarylike structures upon vascular endothelial growth factor (VEGF) A stimulation. The angiogenic outgrowth consisted of endothelial cells, which actively internalized acetylated-low-density lipoprotein, surrounded by pericytes. Computer-assisted quantification of this vascular network demonstrated considerable sensitivity of this assay to several angiogenic inhibitors, including kinase inhibitors and monoclonal antibodies. We also performed targeted gene knockdown on this model by directly infecting explanted umbilical arteries with lentiviruses carrying short-hairpin RNA. Downregulation of VEGFR2 resulted in a significant reduction of the sprouting capability, demonstrating the relevance of human vascular explants for functional genomics studies. Furthermore, a modification of this assay led to development of a 3D model of tumor-driven angiogenesis, in which angiogenic outgrowth was sustained by spheroids of prostate cancer cells in absence of exogenous growth factors. The human arterial ring assay bridges the gap between in vitro endothelial cell and animal model, and is a powerful system for identification of genes and drugs that regulate human angiogenesis. (Blood. 2013;121(21):e129-e137) IntroductionSignificant progress in angiogenesis research has been made after observations that a neoplastic mass does not grow above a few millimeters in diameter without recruiting new vessels. 1,2 Numerous cellular and animal models have been developed to design robust and exhaustive assays able to mechanistically decipher the angiogenic cascade and to validate new antiangiogenic drugs. Endothelial cell (EC) assays can mimic individual steps of the angiogenic cascade, such as cell migration, proliferation, and capillarylike formation. [3][4][5] However, different cell types-such as fibroblasts, pericytes, and smooth muscle cells-play a crucial role during new vessel formation by releasing soluble factors and establishing homotypic and heterotypic interactions. In addition, collective cell migration and extracellular matrix invasion are critical steps in the angiogenic process but are difficult to investigate with cultured cells. 6 Characterization of angiogenesis has therefore been established using mainly animal models. However, although powerful insights into the molecular and cellular mechanisms of angiogenesis have been elucidated by means of transgenic and knockout mouse models, 2,6 the complexity o...

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“…Angiogenesis ex vivo was studied by rat aortic ring assay (Kruger et al, 2000). Briefly, a 48-well plate was first covered with Matrigel (120 μl) and incubated for 30 min at 37˚C.…”

Section: Ex Vivo Rat Aortic Sprouting Assaymentioning

confidence: 99%

“…To verify the anti-angiogenic effect of RCME ex vivo, we employed the rat aortic ring sprouting assay (Kruger et al, 2000). The rat aortic ring sprouting assay is a widely used ex vivo anti-angiogenic model that mimics several stages of angiogenesis including vascular endothelial cell proliferation, migration and tube formation.…”

Section: Inhibition Of Vegf-induced Vessel Sprouting Ex Vivo By Rcmementioning

confidence: 99%

Novel Anti-Angiogenic Activity in Rubus coreanus Miquel Water Extract Suppresses VEGF-Induced Angiogenesis

Kim¹,

Kim²,

Kim³

2014

BSL

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Vascular endothelial growth factor (VEGF) is a key factor involved in the induction of angiogenesis and has become an attractive target for anti-angiogenesis therapies. The purpose of this study was to elucidate the anti-angiogenic activity of Rubus coreanus Miquel water extract (RCME). Rubus coreanus Miquel has long been employed as a traditional medicine, and recent studies have demonstrated that it has measureable biological activities. Thus, we investigated for the first time the effect of RCME on angiogenesis and its underlying signaling pathways. The effects of RCME were tested on in vitro models of angiogenesis, namely, proliferation, migration, invasion and tube formation of human umbilical vein endothelial cells as well as an ex vivo model of vessel sprouting from the rat aorta in response to VEGF. We observed that VEGF-induced angiogenesis was strongly suppressed by RCME treatment compared to that of the control group. Moreover, we found that RCME inhibited VEGF-induced activation of matrix metalloproteinases and phosphorylation of extracellular signal-regulated kinase and p38, and also effectively inhibited phosphorylation of VEGF receptor 2. These results indicated that RCME inhibits angiogenesis by suppressing phosphorylation of the VEGF receptor and may be useful for the treatment of angiogenesis-dependent diseases such as cancer and diabetic retinopathy.

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Endostatin Inhibits Microvessel Formation in the ex Vivo Rat Aortic Ring Angiogenesis Assay

Cited by 75 publications

References 17 publications

Effect of Angiogenic and Antiangiogenic Compounds on the Outgrowth of Capillary Structures from Fetal Mouse Bone Explants

Effect of Angiogenic and Antiangiogenic Compounds on the Outgrowth of Capillary Structures from Fetal Mouse Bone Explants

Modeling human tumor angiogenesis in a three-dimensional culture system

Novel Anti-Angiogenic Activity in Rubus coreanus Miquel Water Extract Suppresses VEGF-Induced Angiogenesis

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